Fluorescent resonance energy transfer: A tool for probing molecular cell-biomaterial interactions in three dimensions

Abstract

The current paradigm in designing biomaterials is to optimize material chemical and physical parameters based on correlations between these parameters and downstream biological responses, whether in vitro or in vivo. Extensive developments in molecular design of biomaterials have facilitated identification of several biophysical and biochemical variables (e.g. adhesion peptide density, substrate elastic modulus) as being critical to cell response. However, these empirical observations do not indicate whether different parameters elicit cell responses by modulating redundant variables of the cell-material interface (e.g. number of cell-material bonds, cell-matrix mechanics). Recently, fluorescence resonance energy transfer (FRET) has been applied to quantitatively analyze parameters of the cell-material interface for both two- and three-dimensional adhesion substrates. Tools based on FRET have been utilized to quantify several parameters of the cell-material interface relevant to cell response, including molecular changes in matrix proteins induced by interactions both with surfaces and cells, the number of bonds between integrins and their adhesion ligands, and changes in the crosslink density of hydrogel synthetic extracellular matrix analogs. As such techniques allow both dynamic and 3-D analyses they will be useful to quantitatively relate downstream cellular responses (e.g. gene expression) to the composition of this interface. Such understanding will allow bioengineers to fully exploit the potential of biomaterials engineered on the molecular scale, by optimizing material chemical and physical properties to a measurable set of interfacial parameters known to elicit a predictable response in a specific cell population. This will facilitate the rational design of complex, multi-functional biomaterials used as model systems for studying diseases or for clinical applications.

Figure 1. Biophysical and Biochemical Variables Affecting Cell Phenotype

Cell responses (e.g. proliferation, gene expression) are modulated by the interface between cell and ECM, which is affected by the presentation of adhesion ligands and the physical properties of the matrix (e.g. compliance). However, cell responses are also affected by soluble chemical factors (e.g. cytokines, growth factors), physical forces (e.g. cyclic strain) and signals from other cells, which may be mechanical or chemical (e.g. paracrine growth factors) in nature. Signals from one source (e.g. soluble chemicals) influence the manner in which signals from other sources (e.g. ECM) are integrated by cells, further complicating correlations between a single material chemical or physical parameter and complex cellular responses in vitro or in vivo.

Figure 2. Controlling Peptide Presentation Variables with Materials Chemistry

2.a: Adhesion epitope can be varied by changing the amino acid sequence of the biomimetic peptide (e.g. RGD vs. YGSIR). 2.b: Peptide structure can also be varied during synthesis, for example to present the same peptide sequence as either a linear or cyclic peptide. The cyclic peptide more closely resembles the conformation of the RGD sequence found in FN. 2.c: The nanoscale organization of the peptide can be isotropic, as shown on the left, or anisotropic, such that the adhesion epitopes (e.g. RGD) is presented in concentrated clusters rather than evenly distributed on the nanoscale. Anisotropic ligand presentation can accomplished by mixing a multivalent polymer attached to multiple epitopes with unmodified polymer. Presenting peptides in an anisotropic fashion allows one to decouple the nanoscale spacing between individual peptides and their microscale density.

Figure 3. Fluorescent Resonance Energy Transfer as a probe for cell-material interactions

3.a: Mammalian cells adhere to alginate sECM exclusively through biomimetic RGD peptides (DIC image). 3.b–c: RGD peptides are separately coupled to donor (e.g. carboxyfluorescein) or acceptor (e.g. tetramethylrhodamine) succinimidyl esters via a lysine residue on the peptide. The solutions of alginate are mixed at an equal ratio and crosslinked. Cellular clustering of fluorophore-coupled RGD peptides presented by alginate sECM can then be monitored by FRET, through reduction in the emission of the green donor (3.b) and corresponding increase in emission of the acceptor (3.c) given excitation of the donor. The donor emission was estimated by sub-dividing the image into a grid and quantifying the number of pixels (per grid subunit) in the green emission channel exceeding an intensity threshold using ImageJ; the degree of energy transfer was calculated by comparing the estimate of donor emission to the donor emission from a control that lacked tetramethylrhodamine tagged RGD according to Equation 1.5. Regions of cells with the greatest degree of contractility (e.g. edges) were found to have the highest degree of energy transfer. The relative force used to cluster peptides can be estimated from the FRET measurement assuming that the mean initial distance between RGD peptides on separate alginate chains is equal to the radius of gyration of alginate in solution (Kong 2005a). 3.d–e: Integrin-RGD bond formation for cells encapsulated into three-dimensional RGD-modified alginate sECM can be monitored via energy transfer from a membrane-embedded fluorescein-tagged lipid analog (donor) and fluorophore (tetramethylrhodamine) attached to RGD (acceptor). When the acceptor is absent, there is minimal emission of the acceptor (3.d). Energy transfer is also inhibited if integrin receptors are saturated with unlabelled, soluble RGD prior to encapsulation, confirming that energy transfer is specific to RGD-integrin bonds (Kong 2006). In contrast, when the acceptor is present, there is marked reduction in green emission and corresponding increase in red emission at the cell-material interface (3.e) (unpublished data).

Figure 1.1. Indications for and Observations During FRET

1.1a: Conditions required for FRET: overlap of the emission spectrum of the donor (“D”) and excitation spectrum of the acceptor (“A”) described by overlap interval J(λ). The overlap integral shown is for the fluorescein-rhodamine pair. 1.1b: Qualitative results of FRET are shown: as the distance r separating the donor and acceptor is decreased, the relative emission of the donor decreases while the emission of the acceptor increases.